Gene encoding interleukin-2 polypeptide, recombinant DNA carrying the gene, a living cell line possessing the recombinant DNA and method for producing interleukin-2 using the cell

ABSTRACT

A gene coded for a polypeptide which possesses interleukin-2 is isolated, and connected with a vector DNA which is capable of replicating in a procaryotic or eucaryotic cell at a position downstream of a promoter gene in the vector obtaining a recombinant DNA, with which the cell is transformed to produce interleukin-2.

This application is a continuation of application Ser. No. 07/814,049,filed Dec. 26, 1991, which issued as U.S. Pat. No. 5,260,868, which is acontinuation of application Ser. No. 07/332,364, filed Apr. 3, 1989,abandoned which is a continuation of application Ser. No. 07/036,309,filed Apr. 7, 1987, abandoned, which is a continuation of applicationSer. No. 06/463,496, filed Feb. 3, 1983, which issued as U.S. Pat. No.4,738,927.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a gene, especially a cloned gene codingfor a interleukin-2 polypeptide, recombinant DNA carrying the gene, aliving cell line possessing the recombinant DNA and a method forproducing interleukin-2 using the cell line.

2. Brief Description of the Prior Art

Interleukin 2 (hereinafter referred to as "IL-2"), fomerly referred toas T cell growth factor, is a soluble protein (generally known as"lymphokine"), and is produced from T cells activated with a lectin oran antigen (Morgan, D. A., et al., Science, 193, 1007-1008 (1976),Gillis, S. et al., J. Immunol., 120, 2027-2033 (1978). Interleukin 2(IL-2) is capable of modulating lymphocyte reactivity and promoting thein vitro long-term culture of antigen specific effector T-lymphocytes(Gillis, S. et al., Nature 268, 154-156 (1977)). IL-2 is also known tomanifest other relevant biological activities such as enhancement ofthymocyte mitogenesis (Chen, B. M. et al., Cell. Immunol., 22, 211-224,(1977), Shaw, J. et al., J. Immunol. 120, 1967-1973, (1978)), inductionof cytotoxic T cell reactivity (Wagner, H. et al., Nature, .284,278-280, (1980)) and anti-SRBC plaque forming cell responses (Gillis, S.et al., J. Exp. Med., 149, 1960-968, (1979)) in cultures of nude mousespleen cells. Accordingly, this lymphocyte regulatory substance isuseful in potentiating humoral and cellular immune responses and inrestoring immune deficient state to a normal humoral and cellular immunestate. These identified immunological activities of IL-2 stronglyindicate that IL-2 is useful for medical immunotherapy againstimmunological disorders including neoplastic diseases, bacterial orviral infections, immune deficient diseases, autoimmune diseasesetc.(Papermaster, B. et al., Adv. Immunopharm., 507, (1980)). Likeinteferons, IL-2 has been shown to augment natural killer cell activity,suggesting a potential use in the treatment of neoplastic diseases.Furthermore, IL-2 enables the maintenance of cultures of functionalmonoclonal T cells and hence appears to play a key role in the studyingof the molecular nature of T cell differentiation, and of the mechanismof differentiated T cell functions as well as the mechanism of T cellantigen receptors. It is also useful for producing,by long termculturing of monoclonal T cell, many other T cell derived lymphokines,which are useful in a wide range of fields. In addition, IL-2 productionand the response of lymphocytes to IL-2 can be important parameters ofimmunological functions which are useful in the clinical diagnosis ofaberrant immunity.

IL-2 has been produced in the prior art by stimulating mouse, rat orhuman lymphocytes with a mitogen (Gillis. S. et al., Nature, 268,154-156, 1977, Farrar, J. et al., J. Immunol., 121, 1353-1360,(1978),Gillis, S. et al., J. Immunol., 120, 2027-2033, 1978,)). By stimulatinghuman peripheral blood mononuclear lymphocytes with a mitogen (Gillis.S. et al., J. Immunol., 124, 1954-1962, (1980)). Gillis et al. reportedthe preparation of murine IL-2 from murine T cell lymphoma cell line(Gillis, S. et al, J. Immunol., 125, 2570-2578 (1980)) and thepreparation of human IL-2 from a human leukemia cell line (Gillis, S. etal., J. Exp. Med., 152, 1709-1719, (1980)).

The above noted articles by Gillis et. al. discuss the method ofproducing human IL-2 from mitogen-stimulated human T cell leukemia cellline by cell culture methods. However, such a technique results inundesirably low concentrations of human IL-2, and necessiates complexpurification procedures to obtain even small amounts of IL-2 from a hugevolumes of culture media. Moreover, since the human T cell leukemia celLlines produce trace amounts of many other biologically active substanceswhich are analogous to human IL-2, significant difficulties areencountered in isolating IL-2 from these other immunologically activemolecules, or in isolating IL-2 from the occasionally present toxiclectins.

As an alternative approach it would seem to be desirable to userecombinant DNA (DNA is an abbreviation for deoxyribonucleic acid)techniques as are used in the production of other biologically activehuman proteins, such as interferons, (Gray, P. W. et al., Nature, 295,503-508, (1981), Nagata, S., et. al., Nature, 284, 316-320, (1980),Taniguchi, T. et. al., Gene, 10, 11-15, (1980)) to produce IL-2. Howeverto date, attempts at the production of IL-2, by recombinant DNAtechniques have not been successful. For instance, it was reported in"NIKKEI BIOTECHNOLOGY (Japan), No. 19, Jul. 5, 1982 that attempts toconstruct IL-2-producing organisms by recombinant DNA were unsuccessful,probably due to the fact that the gene coding for IL-2 polypeptide hadnot yet been cloned.

A need therefore, continues to exist for a cloned gene, coded forinterleukin-2, and for DNA produced recombinantly which carries thegene. A need also continues to exist for a living cell line whichpossesses the recombinantly produced DNA, and for a method of producinginterleukin-2 using the cell line.

SUMMARY OF THE INVENTION

These and other objects of the present invention which will hereinafterbecome more readily apparent from the following description have beenattained by providing:

A cloned gene coded for a polypeptide which possesses the activity ofIL-2, and by providing:

A DNA, produced recombinantly which comprises a gene coded for apolypeptide possesses the activity of IL-2, and a vector DNA capable ofreplicating in a procaryotic or eucaryotic cell, the coding sequence ofthe said gene being located at a position down-stream of a promotersequence.

Further in accordance with the present invention, procaryotic oreucaryotic cell lines are provided which have been transformed toproduce IL-2 with the above said DNA, vector DNA and coded gene. DNAcapable of replicating in the cell; the coding sequence of said the genebeing located at a position downstream of a promoter sequence.

In accordance with the present invention, IL-2 is produced byaerobically culturing a medium containing a eucaryotic or procaryoticcell line which has been transformed to produce IL-2 with a DNA whichhas been recombinantly modified by invention of a gene coded to producewhich possesses the activity of IL-2, and, by insertion of a vector DNA,which is capable of replicating in the cell; the coding sequence of saidgene being located at a position downstream of a promoter sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily attained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows a restriction endonuclease cleavage map of a cloned genecoded to produce a polypeptide which possesses the activity of IL-2(hereinafter referred to as "IL-2 polypeptide").

FIG. 2(a) shows the base sequence of the cloned gene. FIG. 2(b) showsAmino Acid Sequence I, and Amino Acid Sequences II and III, of thepolypeptides which possess IL-2 activity.

FIG. 3 is a flow chart showing the construction of a recombinant DNA(pCEIL-2), in which the coded gene is inserted.

FIG. 4 shows the plasmid vector pTrS-3.

FIGS. 5(a), 5(b) and 5(c) are flow charts showing the construction ofrecombinant DNAs (pTIL 2-22, pTIL2-21, pTIL2-14 and pTIL2-15) usingpTrS-3 as a vector.

FIG. 6 is a flow chart showing the construction of a recombinant DNA(pKIL2-21) using pKT218 as a vector.

FIG. 7 is a flow chart showing the construction of a recombinant DNA(pTuIL2-22) using pTUBlP-5 as a vector.

FIG. 8 is vector DNAs which are capable of replicating in a cell ofSaccharomvces cereviceae.

In the Figures, "A", "G", "C" and "T" represent deoxyadenylic acid,deoxyguanylic acid, deoxycytidylic acid and thymidylic acid,respectively.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENT

The cloned gene, coded for an IL-2 polypeptide, may be obtained bytranscription of messenger RNA (mRNA; "RNA" is an abbreviation forribonucleic acid) corresponding to IL-2 (hereinafter referred to as"IL-2 mRNA"), originating from a mammalian cell which is characterizedby the capability of producing a polypeptide which possesses IL-2activity, to a complementary DNA (cDNA). The single stranded cDNA(ss-cDNA) obtained can be converted into a double stranded cDNA(ds-cDNA).

The mRNA used as a template for the preparation of cDNA can beconventionally separated from a mammalian cell capable of producing IL-2polypeptide. The separated RNA is polyadenylated (Gillis, et al.,Immunological Rew., 63, 167-209 (1982)), and the polyadenylated RNA canbe fractionated by, for example, centrifugation on a sucrose densitygradient as a sediment of 11 to 12S . Occasionally mRNA of 13S willdemonstrate IL-2 mRNA activity, and in those instances, it is presumedthat the mRNA is in an aggregated form of 11 to 12S mRNA.

The mammalian cells capable of producing IL-2 which are the source ofmRNA of the present invention, may be T-lymphocytes, such as peripheralblood mononuclear cells, tonsil cells, spleen cells or the like, whichare operationally obtainable from mammals. The cells may beconventionally pretreated such as with a nylon column,antiserum-complement, density gradient fractionation, multiple enzymetreatment such as a combination of neuraminidase and galactose oxidase,by x-ray irradiation or with trypsin to confer the cells with the IL-2productivity or to increase the IL-2 activity. Also cloned Tlymphocytes, obtained from the said mammalian cells after cultivation inthe presence of T cell growth factor, may be also used as a source ofmRNA and is the preferred T-lymphocytes. Transformed lymphocyte celllines such as T lymphocytes derived from leukemia or lymphoma cell lineper se or from their derivatives obtained by pretreatment or mutation bythe methods mentioned above, or the cloned transformed cell lines arepreferred as sources of the MRNA. Evidently, cloned cells line usuallycontain larger amounts of IL-2 mRNA as compared to parental bulk celllines. T cell hybridomas, obtained by fusion of the lymphocyte derivedcells mentioned above and tumor cell lines, such as CEM, Molt 4F, andBW5147, are also preferred mammalian cell lines for use in thisinvention. In such instance the lymphocyte derived cell lines include(1) constitutive producers of IL-2 and (2) those which are producers ofIL-2 only in the presence of a mitogen introduced into the culture,either in the absence or presence of other IL-2 productionco-stimulatory cells.

In order to generate IL-2 MRNA in constitutive IL-2 producer cells, theconstitutive IL-2 producer cells are cultured under conditions commonlyknown in the field of cell culture. For the generation of the mRNA incells producing IL-2 only in the presence of mitogen, cultured cells arewashed extensively with culture medium and resuspended in a culturemedium, such as Rosewell Park Memorial Institute 1640 (hereinafter "RPMI1640"), Dulbecco Modified Eagle Medium (hereinafter "DMEM") or inClick's medium, which may or may not contain serum. These culture mediamay be supplemented with various additives, such as penicillin,streptomycin or other antibiotics, or with fresh L-glutamine, Hepesbuffer and sodium bicarbonate in a concentrations as are generally usedin the field of cell culture. The preferred cell density may be from 0.5to 4×10⁶ cells/ml. To induce activation of the mRNA and production ofIL-2, appropriate stimulants are added. Suitable such stimulants includemitogens, neuraminidase, galactose oxidase, zinc derivatives such aszinc chloride, or lymphocyte activating substances originated frommicroorganisms, such as protein A, streptolysin-O. The stimulated cellsare recovered and washed. The co-presence of macrophages or dendriticcells during the mitogen stimulation may also activate the mRNA, or mayincrease the amount of the activated mDNA. Likewise the co-presence ofcell lines derived from B lymphocytes or B lymphocyte lines, such asRaji, Daudi, K562, and BALL-1 may activate the mRNA or increase theamount of activated MRNA.

To propagate the mammalian cells, they are maintained in an in vitrocell culture or in histocompatibility matched animals, under normalconditions. When in vitro culture maintenance is used to prepare thesource of mRNA, the cells may be grown in any of the culturing media aswere previously found to foster growth of T cells. These culture mediamay or may not be supplemented with mammal serum, serum component orserum albumin. The culture period for the activation of the mRNA willcorrespond to the period necessary for the activation of cells togenerate the mRNA. This period usually coincides with the time needed tostart the excretion of IL-2 into the culture medium. The preferredperiod may be from 3 to 12 hours after addition of a stimulant, such asa mitogen. Undue prolongation of the culture period may. occasionallyresult in the decomposition of the generated IL-2 mRNA. During thecourse of the activation of IL-2 producing cells, phorbol esters, suchas PMA or TPA may preferably utilized in a concentration from 10 to 50ng/ml to boost the level of activation.

The above described process for activation of IL-2 mRNA may be carriedout at temperatures ranging from 32° to 38° C. in a humidifiedatmosphere and in a pH of approximately 7.0 to 7.4.

The procedures to obtain and culture mammalian cells capable ofproducing IL-2 will now be explained.

(1) Acquisition of constitutively IL-2 producing cell line.

Jurkat cell line of human leukemic T cell (freely available from FredHutchinson Cancer Institue, Seattle, United States, Salk Institute, SanDiego, United States, German Cancer Center, Heidelberg, West Germany) issuspended in Click's medium at a cell density of 1×10⁶ cells/ml and8×10³ R x-ray is irradiated at irradiation rate of 150 R/min. Thereafter0.1 cells of the thus irradiated cells per 200 μl of medium areinoculated into Click's medium containing 5% FCS in 96 well flat-bottommicroplates (Falcon 3072) and cultured for 3 weeks at 37° C. in 5% CO₂incubator (cloning by limiting dilution method). The grown viable cellsare transferred into 24 well culture plate (Nunc) before the cell layerbecomes confluent and is further cultured for 5 days. The grown cellsare further cultured in serum and serum albumin free synthetic culturemedium for about two days at a initial cell density of between 1-2×10⁶/ml. The culture supernatant is harvested by centrifugation and filteredwith 0.22 millipore filter paper to clear off debris and to sterilizethe supernatant, and then x-ray treated mutants capable of producingIL-2 constitutively are selected and cloned by measuring the IL-2activity present in the supernatant.

(2) Acquisition of IL-2 producer cell from human peripheral bloodmononuclear cells.

Human peripheral blood is harvested and peripheral blood lymphocytes(hereinafter "PBL") are isolated by density gradient centrifugation onFicoll-Hypaque. The PBL is inoculated in 2 ml of Click's mediumcontaining 5% FCS at a cell density of 1×10⁶ cells/ml in 24 well Nuncculture plate together with 100 μl of 5 μg/ml ofphytohemmaglutinin-M(Gibco)(pHA), and cultured for 48 hrs under theconditions described above. The cells are washed and inoculated again in1 ml of Click's medium at a cell density of 1×10⁵ cells/ml together with1 ml of a conditioned medium which has been prepared from humansplenocytes stimulated by 2.5 μg/ml of concanavalin A (hereinafter "ConA") for 48 hrs, and the culture medium containing 50% conditioned mediumis exchanged in every three days to get long term culture of human Tlymphocytes from PBL. The thus prepared long term cultured human Tlymphocytes are cloned by the limiting dilution method as describedabove, in the presence of human splenocytes derived conditioned mediumand the cell clones are propagated similarly. Thereafter cloned human Tlymphocytes are inoculated in 1 ml of RPMI 1640 at a cell density of1×10⁶ cells/ml in 24 well Nunc culture plate in the presence of 10 μg/mlof PHA and cultured for 24 hrs at 37° C. in 7.5% CO₂ incubator. Thesupernatants of the culture liquid are harvested, centrifuged, filteredthrough a 0.22μ millipore filter and assayed for IL-2 activity tospecify the IL-2 producing human normal T lymphocytes clones.

(3) Acquisition of malignant cell line derived from human lymphocytescapable of producing IL-2 in the presence of mitogen.

Jurkat cell line or cloned cell lines such as Jurkat 111 obtained by thelimiting dilution method described above are able to produce from 10 to4,000 units/ml of IL-2 when cultured for 24 hours in a serum freesynthetic medium described previously or in RPMI 1640 containing 1-2%mammalian serum in the presence of a mitogen such as 10 μg/ml Con A or2.5 μg/ml PHA. These malignant human cell lines also produce IL-2 whencultured in the presence of zinc chloride, protein A or picibanil.

(4) Acquisition of cells capable of producing IL-2 in the co-presence ofa mitogen and other co-stimulatory cells or co-stimulatory solublefactors.

Human malignant cell line Molt 4F and some cloned cell lines such asJurkat J99, obtained according to the limiting dilution method, do notproduce IL-2 even when cultured for 24 to 72 hours in the presence oflectins or mitogens in any concentration. However, these cells becomeable to produce IL-2 in significant amount (10-100 μ/ml) during cultureperiod of 24 hours at 37° C., when cocultured with 5-10 μ/ml interleukin1, one of monokines, or with 50% number of K562 or Raji cells.

The extraction of IL-2 MRNA from cells activated by the manner asmentioned above is carried out according to the conventional well knownprocedures, irrespective of the difference of cell sources. Forinstance, cells are partially or completely disrupted by addition of adetergent such as NP-40, SDS, Triton-X and deoxycholic acid or bymechanical homogenization or freeze-thawing. To prevent degradation ofRNA by ribonuclease during extraction of mRNA, it is preferred to addRNase inhibitors such as heparin, polyvinylsulfate, bentonite, macaroid,diethylpyrocarbonate or vanadyl complex. IL-2 MRNA can be obtained fromprecipitated polysome in the IL-2 biosyntheis, which is precipitatedwith anti-IL-2 antibody by extracting with a detergent.

The poly A-containing maNA can be fractionated or concentrated by anyconventional manner, such as by affinity chromatography or batchabsorption on oligo dT-cellulose, poly U-sepharose of sepharose 2B,sucrose density gradient centrifugation or by agarose gelelectrophresis.

The MRNA fractions are then assayed for IL-2 mRNA activity by testingbiological activities of proteins translated from the MRNA fractions orby identifying the translated protein using monoclonal antibody againstthe IL-2 peptide. For instance mRNA is usually translated into thecorresponding protein by microinjection into frog (Xenopus laevis) egg(Gurdon, J. B., et al., Nature, 233, 177-182 (1972)) or by employing themRNA dependent reticulolysate or wheat germ translation cell freesystems.

The activity of IL-2 may be ascertained by the microassay procedureprincipally discussed by Gillis et. al (Gillis. S., et al., J. Immunol.,120, 2027-2033 (1978)). The assay monitors the IL-2 dependent cellularproliferation of a cytotoxic T lymphocyte cell lines (hereinafter"CTLL") generated according to the methods described by Gillis et al.,That is, 4×10³ CTLL cells are inoculated into 100 μl of RPM1 1640 mediumcontaining 2% FCS in 96 well flat-bottomed microplates together with 100μl of the serially diluted translation products. After 20 hoursincubation at 37° C. in 5% CO₂ incubator, cells are pulsed for 4 hourswith 0.5 μCi of ³ H-TdR, harvested onto glass fibre strips with the aidof an automated cell harvester and then the incorporated radioactivityis measured by liquid scintillation counting. By these assay procedures,the CTLL cells cultured in the presence of IL-2 were found toincorporate ³ H-TdR in a dose dependent manner resulting in the definitecalculation of the amount of IL-2 contained in test samples.

IL-2 possesses the activity to promote the proliferation of Tlymphocytes, which enables the measurement of IL-2 activity using anindex of T cell growth activity. That is, five CTLL cells aretransferred into 100 μl of DMES containing 2% FCS in 96 wellflat-bottomed microplates together with 100 μl of the serially dilutedtranslation products. After 72 to 96 hours incubation at 37° C. in a 5%CO₂ incubator, the number of cells grown and activated is counted undermicroscopy. As an positive external control group, 100 units/ml, 10units/ml of IL-2 are added and the IL-2 activity of the test sample iscalculated in comparison with the number of grown viable cells in thesecontrol groups.

The thus obtained IL-2 mRNA from the most active fraction is used as atemplate to synthesize ds-cDNA and the ds-cDNA is connected with avector DNA.Synthesis of cDNA is carried out by conventional procedures.

At first ss-cDNA which is complementary to mRNA is prepared in thepresence of dATP, dCTP, dTTP employing reverse transcriptase and usingmRNA as a template and oligo-dT as a primer. The template mRNA is thenremoved by alkaline treatment and ds-cDNA is achieved by employingreverse transcriptase or DNA polymerase and using the above synthesizedss-cDNA as a template.

DNA produced recombinantly is prepared from the ds-cDNA thus obtainedand a vector DNA containing replicon capable of replicating ineucaryotic or procaryotic cells. The recombinant DNA is thereafterincorporated into the host cells.

The ds-cDNA and a vector DNA capable of propagating in eucaryotic orprocaryotic cells are, prior to ligation, modified by various proceduressuch as exonuclease treatment, addition of chemically synthesized DNApieces and G, C-tailing to give ligatable termini to the ends of theds-cDNA and the vector DNA. Ligation of the ligatable DNAs is performedby, for example, T₄ -phage DNA ligase in the presence of ATP.

With the recombinant DNA thus obtained, living cells are transformed toamplify the cloned cDNA or to produce IL-2 polypeptide.

Suitable eucaryotic host organisms, which are usually used forproduction of IL-2, include vertebrates, yeasts and so on. For instance,monkey cells, e.g. CV-1 cells, transformed by an origin defective mutantof SV-40 and expressing the SV-40 large T antigen (COS cells) asdiscussed by Y. Gluzman (Cell, 23, 175-182, 1981), mouse derived cellsdiscussed by Ohno, S. and Taniguchi, T. (Nucleic Acids Research, 10,967-977, (1982)), and yeast host-vector systems applied for theexpression of IFN gene, discussed by R. Hitzeman et al. (Nature, 293,717-722, (1981)) may be used. Suitable procaryotic host organismsinclude Escherichia coli, Bacillus subtilis and so on. For theamplification of DNA in host organisms, it may be preferred to use E.coli as a host, however other hosts can also be employed.

Suitable vectors used for E. coli include EK type plasmid vector(stringent type): pSC101, pRK353, pRK646, pRK248, pDF41 etc., EK typeplasmid vector (relaxed type): ColE1, pVH51, pAC105, RSF2124, pCR1,pMB9, pBR313, pBR322, pBR324, pBR325, pBR327, pBR328, pKY2289, pKY2700,pKN80, pKC7, pKB158, pMK2004, pACYC1, pACYC184, dul etc. λgt type phagevector: λgt. λc, λgt. λB, λWES, λC, λWES. λB, λZJvir., λB', λALO, λB,λWES. Ts622, λDam etc.. In general pBR322 has been frequently used as avector for E. coli and in that instance the best cloning sites are thePst I and EcoRI sites.

Transformation of the host cell with the recombinant DNA may be carriedout by conventionally used manner as follows:

Where the host is of procaryote such as E. coli, competent cells whichare capable of DNA uptake are prepared from cells harvested afterexponential growth phase and subsquently treated by the CaCl₂ -method bywell known procedures. When MgCl₂ or RbCl exists in the transformationreaction medium, the transformation efficiency increases. Transformationcan be also performed after forming a protcplast of the host cell.

Where the host used is an eucaryote, transfection method or DNA ascalcium phosphate-precipitates,conventional mechanical procedures suchas microinjection, insertion of a plasmid encapsulated in red blood cellhosts or in liposomes, treatment of cells with agents such aslysophosphatidylcholine, or use of virus vectors, or the like may beused.

Cells possessing IL-2 gene can be isolated after the transformation, byeither of the following two ways.

(1) In the plus-minus method, partially purified IL-2 MRNA is obtainedby sucrose density gradient centrifugation of mRNAs extracted frommitogen activated mammalian cells as 11 to 12s sediment and then 32p-radiolabelled ss-cDNA is synthesized using the partially purified mRNAas a template. After removal of the template mRNA by alkaline treatment,isolated cDNA is hybridized with partially purified 11 to 12s mRNAextracted from mitogen non activated mammalian cells. Thereafternonhybridized and hybrid forming cDNA are fractionated onhydroxylapatite column chromatography. The non hybridized cDNA andhybridized cDNA are tentatively called probe A and probe B,respectively. Transformants are grown on two nitrocellulose filters inquite the same way: and the DNA of the cells is fixed on the filterpaper by alkaline treatment. Probe A and Probe B are respectivelyhybridized with the DNA on two different filter papers and thereafterautoradiography assay is carried out to select the transformants whichreact positively to probe A (plus), but react weakly or do not at all toprobe B (minus)(Taniguchi et al., Proc. Jpn. Acad., v 155B 464-469,1979).

(2) The second method consists of dividing, for example,1,000 to 10,000transformant clones into several tens or several hundreds of clonegroups. The divided clone groups are respectively cultured byconventional means to obtain plasmid DNAs. Thereafter these plasmid DNAsare converted into ss-cDNAs, for example, by heat denaturation, and thess-cDNAs obtained are fixed onto nitrocellulose filter papers to achievethe hybridization of mRNA complementary to the fixed DNAs and preparedfrom mammalian cells including activated IL-2 mRNA. Alternatively, mRNAscontaining IL-2 MRNA are hybridized with heat denaturated plasmid DNAsand then DNA-mRNA hybrid is fixed onto nitrocellulose filter papers.These filter papers are then washed with low salt concentration buffer,such as 1 mM HEPES, or with 10 mM NaCl, and mRNA adsorbed on filterpaper is extracted by treatment with a solution containing 0.5 mM EDTAand 0.1% SDS solution for e.g. 1 min. at 95° C. Purified mRNA isrecovered by elution through oligo dT-cellulose column chromatography.Thereafter, the MRNA is translated into protein by microinjection intoXenopus laevis egg to ascertain IL-2 activity, or the MRNA is translatedinto a protein using the mRNA dependent reticulocyte or wheat germ invitro cell free translation system, to analyse IL-2 activity usinganti-IL-2 antibody. According to these procedures, tre group in whichthe presence of IL-2 activity was detected was further dividedrepeatedly into groups consisting of smaller number of transformantclones until a single clone possessing IL-2 DNA is specified.

To obtain cDNA codina for IL-2 polypeptide from the IL-2 producingtransformant, the recombinant DNA in the transformant is separated andcleaved with a restriction endonuclease. From the DNA fragments formedby the cleaving, the insert cDNA fraction is separated.

The complete nucleotide sequence of the PstI DNA insert coding for IL-2polypeptides from the recombinant DNA of pIL2-50A was determined by theprocedure of Maxam and Gilbert (Meth. Enzym. 65. 499-560, (1980)) and bythe dideoxynucleotide chain termination method (Smith, A. J. M. Meth.Enzym. 65, 560-580 (1980)).

The restriction endonuclease cleavage map of the cDNA insert and basesequence of the insert are shown in FIG. 1, and FIG. 2(a) in which thecDNA has sites cleaved with restriction endonuclease of BstNI, XbaI andBstNI in this order, respectively.

The DNA sequence of the insert contains a single large open readingframe. The first ATG sequence, which usually serves as the initiationsequence in eukaryotes (Kozak, M. Cell, 15, 1109-1123 (1978)), is foundat nucleotides 48-50 from the 5' end. This ATG is followed by 152 codonsbefore the termination tripLet TGA is encountered at nucleotides 507 to509. A stretch of A residues corresponding to the 3'-poly (A) terminusof the MRNA is found at the end of the cDNA and this is preceeded by thehexanucleotide AATAAA (position 771-776) which is usually found in mosteukaryotic mRNAs (Proudfoot, N. J. and Brownlee, C. G., Nature 263,211-214, (1976)).

The amino acid sequence, for which the cDNA codes, could be deduced asshown in FIG. 2(b) (Amino Acid sequence I), and the polypeptide of theamino acid sequence I consists of 153 amino acids and its molecularweight is calculated to be 17631.7 daltons. As has been reported as acommon feature in most of the secretion proteins known to date (Blobel,G. et al., Sym. Soc. exp. Med., 33, 9-36 (1979)), the N-terminal regionof the deduced IL-2 polypeptide is also quite hydrophobic and thisregion probably serves as a signal peptide which is cleaved during thesecretion process of the mature IL-2. Such cleavage occurs eitherbetween Ser and Ala at position 20 and 21 or between Ala and Pro atposition 21 and 22 respectively, forming the polypeptide having aminoacid sequences II and III, since similar cleavage sites have often beenfound in other secretion proteins (Blobel, G. et. al., Symp. Soc, exp.Med. 33, 9-36, (1979)). The mature IL-2 polypeptide would then contain133 or 132 amino acids with the calculated molecular weight being15420.5 daltons or 15349.4 daltons. This value is then compared with thereported value for human IL-2 protein from Jurkat cells (15,000 daltons)(Gillis, S. et al., Immunological Rev., 63, 67-209, (1982)).Additionally, the DNA fragment initiating from CCT codon at position 111to 113 in base sequence, which, therefore, codes for a polypeptideinitiating from Pro at position 22 (Amino Acid Sequence III in FIG.2(b)), was confirmed to express a polypeptide possessing IL-2 activityas shown in Example 5. It is also confirmed that the DNA fragmentinitiating from GCA sequence at position 107 to 110 in the basesequence, which therefore codes for a polypeptide initiating from Ala atposition 21 (Amino Acid Sequence II in FIG. 2(b)) expresses apolypeptide possessing IL-2 activity as shown in Example 8.

It has been known that genes of eukaryotes often show polymorphism forexample in human interferon genes. (Taniguchi et al. Gene 10. 11-15(1980), Ohno & Taniguchi, Proc. Natl. Acad. Sci U.S.A., 77, 5305-5309,(1986); Gray et al., Nature, 295 501-508 (1981)). In some cases,polymorphysm is accompanied with replacement of certain amino acids ofthe protein products and in other cases, the structure of the proteinproduct remains unchanged. In the case of human IL-2 cDNA, another cDNAclone (pIL2-503) in which the A residue at position 503 of pIL2-50A cDNA(FIG. 2) is replaced by a G residue can be detected. Other cDNA cloneswith some base substitution compared to pIL 2-50A cDNA can also beexpected.

As can be understood from the above the genes of present inventioninclude DNA having the base sequence shown in FIG. 2(a), DNAs initiatingfrom ATG sequence at position 48 to 50 and having the sequencial basesfollowing the ATG sequence up to at least ATC sequence at position504-506, DNAs initiating from GCA sequence at position 108-110 andhaving the secuencial bases following the GCA sequence up to at leastthe ATC codon and DNAs initiating from CCT sequence at position 111-113and having the sequencial bases following the CCT sequence up to atleast the ACT sequence. The genes of the present invention also includeDNAs ending at the ACT sequence at position 504 to 506 and initiatingfrom A at position 1, ATG sequence at position 48 to 50, GCA sequence atposition 108 to 110 or CCT sequence at position 111 to 113. The genes ofthe present invention further include DNAs ending at TGA sequence atposition 507 to 509 and initiating from A at position 1, ATG sequence atposition 48 to 50, GCA sequence at position 108 to 110 or CCT sequenceat position 111 to 113. The genes of the present invention furtherinclude DNAs ending at C at position 801 and initiating from A atposition 1, ATG sequence at position 48 to 50, GCA sequence at position108 to 110 or CCT sequence at position 111 to 113. The genes of thepresent invention additionally include DNAs ending with poly (A) andinitiating from ATG codon at position 48-50, GCA sequence at position108-110 or CCT sequence at position 111 to 113. The genes of the presentinvention also include those of which base seauences correspond to AminoAcid Secuence I, II and III. Furthermore, polypeptides deficient in oneor more amino acids in Amino Acid Sequence I, or polypeptides in whichone or more amino acids in Amino Acid Sequence I are replaced with oneor more amino acids may have IL-2 activity. Therefore genes coded forsuch polypeptides are suitable genes for the present invention.Similarly, genes having additive connection of one or more basesequences, capable of expressing one or more amino acids to Amino AcidSequences I, II or III, are sutable in this invention so far as theadditively connected amino acids do not interfere with the action of thepolypeptides in expressing IL-2 activity. Modified additively connectedamino acid region which interfere with the polypeptide function as IL-2,can be used in this invention so far as the additively connected regioncan be easily eliminated. This situation is quite the same for theadditive connection of DNA to the 3'-terminus of genes corresponding toAmino Acid Sequence I, II and III coding additional amino acids atC-terminal of the I, II and III having Amino Acid Sequence I, II and IIIrespectively. Therefore use of genes coded for such polypeptides are tobe considered to be included in the present invention.

Recombinant DNAs which direct the production of IL-2 in living cells canbe constructed by various methods. For example, the coding sequence ofIL-2 cDNA can be inserted in an expression vechcle downstream of thepromoter sequence. Alternatively, a cDNA piece carrying a. promotersequence can be inserted upstream of the IL-2 coding sequence, after orprior to, the insertion of cDNA in the expression vechcle.

Procedures to construct cells procaryotic or eucaryotic which expressthe IL-2 cDNA and-produce IL-2 polypeptide are explained more preciselybelow:

(1) Expression of the IL-2 cDNA in E. coli

In order to express the IL-2 cDNA in E. coli, the cDNA is fused withvarious bacterial promoters and hybrid plasmids containing the cDNAdownstream of the promoters are obtained. The plasmids are transfectedinto, for example, E. coli HB101 strain and bacteria synthesizing aprotein product with human IL-2 activity are cloned. Essentially anykind of bacterial promoter should direct the expression of IL-2 cDNAwhen they are abutted appropriately to the cDNA. Examples of this cDNAexpression are described here.

The cloned cDNA for IL-2 encodes a polypeptide consisting of 153 aminoacids as illustrated in FIG. 2. The N-terminal region corresponding toabout 20 amino acids of this polypeptide is quite hydrophobic and thisis characteristic of most of the secretion proteins. Such a hydrophobicsequence, so-called signal sequence, is cleaved during the secretionprocess. Therefore, mature IL-2 polypeptide should contain less than 153amino acids. It is therefore desiable to express the cDNA portionencoding the mature IL-2 polypeptide but not the portion correspondingto the IL-2 signal sequence.

(i) Construction of an expression plasmid vechcle, pTrS-3, whichincludes E. coli trp promoter, its ribosome binding site (SD sequence)for the leader peptide was previously reported (G. Miozzari andYanofsky, C. J. Bacteriol. 133, 1457-1466,(1978)) and an ATG codonsituated 13 bp downstream of the SD sequence (Nishi et. al., SEIKAGAKU,54, No. 8, 676 (1982)). The plasmid vehicle also contains a single SphIsite just downstream of the ATG intiation sequence (FIG. 4).

To express IL-2 cDNA, the plasmid is first digested by SphI and treatedeither with E. coli DNA polymerase I (Klenow Fragment) or withbacteriophage T4 DNA polymerase I to remove the 3' protruding ends (FIG.(a)). Plasmid pIL2-50A is double digested by PstI and HaiAI, and alarger cDNA fragment is isolated. The DNA is then treated either with E.coli DNA polymerase I (Klenow Fragment) or with bacteriophage T4 DNApolymerase so that the 3' protruding ends are rendered flush. The abovetreated cDNA encodes IL-2 polypeptide of 132 amino acids as shown inFIG. 5(a). This cDNA is then ligated to the pTrS-3 plasmid DNApre-treated as above, such that the ATG initiation codon is abutted tothe CCT (Pro) sequence of the IL-2 cDNA. Thus, a plasmid pTIL2-22 isobtained. The junction between trp promoter sequence and IL-2 cDNAsequence of pTIL2-22 is also illustrated in FIG. 5(a).

The plasmid pTIL2-22 should direct the synthesis in E. coli of an IL-2polypeptide consisting 132 amino acids starting with proline.

(ii) Since it is also possible that the mature IL-2 contains alanine(position 21) as the N-terminal amino acid instead of proline, thefollowing plasmid which direct the synthesis of IL-2 polypeptide,consisting of 133 amino acids, is discussed,

Plasmid pTrS-3 contains a single ClaI site between SD sequence and ATGsequence (FIG. 4). This plasmid is digested by Cla I and Sal I. PlasmidpIL2-50A is partially digested by PstI, treated with E. coli DNApolymerase I and the largest linear DNA is isolated. The DNA was thenligated with a synthetic DNA linker containing a restriction XhoIcleavage site and a clone containing the plasmid pIL2-50A (Xho) in whichthe linker DNA is introduced at the 3' downstream of IL-2 codingsequence is isolated. The plasmid pIL2-50A(Xho) is first digested byHgiAI, treated either with E. coli Klenow Fragment or with T4 DNAplymerase, digested by XhoI and the cDNA fragment is isolated. This cDNAfragment is then ligated with pTrS-3 DNA pretreated with ClaI and SalIand with a synthetic DNA shown in FIG. 5(b). Thus, a plasmid pTIL2-21which should direct the synthesis in E. coli of an IL-2 polypeptideconsisting 133 amino acids starting from alanine can be obtained asillustrated in FIG. 5(b). Similar construction can also be made withoutusing XhoI linker.

(iii) IL-2 polypeptides with different size with different N-terminalamino acid can be produced by using the pTrS-3 expression plasmidvechcle by the following procedure. The cloned IL-2 cDNA in pIL2-50Acontains a sole DdeI site at nucleotide position 81-85. PlasmidpIL2-50A(Xho) is digested by DdeI and the DNA fragment containing thelarger portion or the cDNA is isolated. The fragment should also containDNA or base about 3,000 pairs from pBR322 (FIG. 5(c)). The DNA fragmentis treated by exonuclcease Bal 31 and then digested by XhoI. The abovetreated DNA is then ligated with pTrS-3 which is digested by SphI,treated either with Klenow Fragment or with T4 DNA polymerase and thendigested by SalI as illustrated in FIG. 5(c). The ligated DNA istransfected into E. coli HB101 and bacterial clones expressing humanIL-2 are screened. Those clones should express human IL-2 of varioussizes since the DNA corresponding to the N-terminal region of human IL-2is variably chewed away. Thus pTIL2-14 and pTIL2-15 carrying IL-2 cDNAcould be obtained.

(iv) The IL-2 cDNA can also be expressed by the use of pKT218 (providedby K. Talmage)(Proc. Natl, Acad, Sci, U.S.A., 77, p3369-3373, (1980)).:Plasmid pKT218 is digested by PstI and ligated with an IL-2 cDNA insertobtained by digesting pIL2-50A DNA by HgiAI and PstI (FIG. 6). Theresulting plasmid pKIL2-21 has the sequence at the beginning of theprotein synthesis initiation as shown in FIG. 6. Thus, the plasmidpKIL2-21 should direct the synthesis in E. coli of a fused polypeptideconsisting of 133 amino acids of IL-2 and amino acid of β-lactamase (Thefirst methionine is cleaved off in E. coli).

(v) An expression plasmid pTuB1P-5 in which the promoter sequence fortuf B is inserted into pBR322 was previously constructed (Taniguchi etal.,SEIKAGAKU,53,966,(1981)). The plasmid contains a single ClaI siteand this is located 2 bp downstream of the SD sequence as shown in FIG.7.

Since pTrS-3 also contains a ClaI site between the SD sequence and ATGinitiation sequence, and since this ClaI site is not destroyed duringthe construction of expression plasmid by using pTrS-3 and IL-2 cDNA asdescribed above, it is very simple to replace the bacterial Trp promoterwith that of tufB so that the human IL-2 cDNA is expressed under thecontrol of tufB promoter. For example, pTIL2-22 is digested by ClaI andPvuII and the DNA fragment containing the IL-2 cDNA is isolated. Thisfragment is then ligated with pTUB1P-5 DNA, pre-digested by ClaI andPvuII and a plasmid pTuIL2-22 is constructed as illustrated in FIG. 7.The IL-2 activity could be detected in the extract of E. coli HB101harboring the plasmid pTuIL2-22.

(vi) Similar construction can also be made by using, for example,pTIL2-21 and essentially all expression plasmids which are constructedwith the use of pTrS-3. It is also possible to optimize the distancebetween SD and ATG sequence by digesting, for example, pTuIL2-22 withClaI, removing (or adding) a few base pairs of DNA by Bal 31 or S1 orDNA polymerase I (E. coli) and then re-ligating the plasmid.

(2) Expression of the IL-2 cDNA in yeast

IL-2 cDNA can be also expressed in yeast by inserting the cDNA intoappropriate expression vectors and introducing the product into the hostcells. Various shuttle vectors for the expression of foreign genes inyeast have been reported (Heitzman. R. et al., Nature 293, 717-722(1981); Valenzuela, P. et al., Nature 298, 347-350 (1982); Miyanoshitaet al., Proc. Natl, Acad. Sci. U.S.A. 80, 1-5 (1983)). The vectors arecapable of replicating both in E. coli and in yeast hosts and theycontain promoter sequences of genes of yeast. Essentially all suchexpression vectors can be used for the expression of IL-2 cDNA. It maybe possible to achieve higher levels of IL-2 production by using yeastas compared to use of animal cells or bacteria. An example of human IL-2cDNA expression in yeast is now described.

A yeast E. coli shuttle vectors pAT77 and pAM82 have been described byMiyanoshita et al. (Proc. Natl. Acad. Sci. U.S.A. 80, 1-5. (1983)). Thevector pAM82 is a derivative of pAT77 and both carrying markers of ars 1(Stinchcomb, D. T. et al, Nature 282, 39-43, (1979), 2 μm ori (Broach,J. R. et al. Gene 8, 121-113 (1979)) and leu 2 (Ratzkin, B. et al. Proc.Natl. Acad. Sci. U.S.A. 74, 474-491 (1979)) and the promoter for theyeast acid phosphatase (APase) gene. They also carry a 3.7 kb DNAsegment of pBR322 which contains an ampicillin resistance marker(Ap^(r)) and the origin of replication (FIG. 8). The APase promoter isinducible by shifting a high concentration of phosphate into a lowconcentration in the culture media. In order to express human IL-2 cDNA,pIL2-50A is digested by PstI after treating either by the E. coli KlenowFragment of by T4 DNA polymerase, the cDNA is ligated with pAM82previously digested by XhoI and incubated with the E. coli KlenowFragment to fill in the ends. Hybrid plasmids in which the cDNA codingsequence are downstream of the yeast APase promoter sequence areselected by cloning them in E. coli. The obtained plasmid, pYIL-2a, isintroduced into yeast and, after induction of the APase promoter, IL-2activity in the yeast extract is measured. The plasmid pYIL-2a containsa stretch of GC residues between the yeast promoter and the IL-2 cDNA.It is possible that such a sequence inhibits the expression of IL-2cDNA. In order to overcome this problem, following construction of aplasmid can be made: Plasmid pIL2-50A is digested by PstI and the cDNAinsert is isolated. This cDNA is then treated by T4 DNA polymerase inthe presence of dATP, dGTP and dTTP so that streches of C-residues atthe both ends of the cDNA are chewed off and subsequently treated byNuclease S1, to remove stretches of G-residues. This DNA is ligated withXhoI DNA linker and plasmid pBR322 whose EcoRI site is cleaved andrendered flush by EcoRI and the Klenow Fragment, and the resultingplasmid, pIL2-Xho, is digested by XhoI and the cDNA insert is isolated.The cDNA is then introduced into the single XhoI site of pAM82 and aplasmid containing the IL-2 coding sequence correctly oriented withrespect to the yeast APase promoter is cloned in E. coli. The plasmid,pYIL-2b, is introduced into yeast and, after induction of the APasepromoter, IL-2 activity in the yeast extract is measured.

(3) Expression of the cDNA in mammalian cell

A plasmid which should direct the synthesis of human IL-2 in mammaliancells can be constructed as follows. A plasmid pCE-1 is constructed fromPKCR (O'Hare, K., et al., Proc. Natl. Acad. Sci. U.S.A., 78, 1527-1531,(1981)) and pBR328 (Soberon, X., et al., Gene, 9, 287-305, 1980) by aseries of modification procedures as illustrated in FIG.2, andinitiation sequence ATG of IL-2 gene is connected to the downstream ofthe SV40 early gene. This plasmid contains a single PstI site justdownstream of the SV40 early promoter and upstream of the part of therabbit β-globin chromosomal gene containing one intron. The plasmid alsocontains the replication origin of SV40 as well as the polyadenylationsite for the early gene. Thus a plasmid pCEIL-2, in which the IL-2structural gene should be transcribed from the early promoter of SV40 inappropriate host cells, is obtained (FIG. 2).

This plasmid is digested by Hhal and then introduced by DNA transfectioninto the transformed monkey cell line COS-7 which allows replication ofDNA containing SV40 origin sequenccs. It appears to be important todigest the plasmid by Hhal before transfection for the efficientexpresssion of cDNA since sequences which could hamper replication ofthe transfected DNA in COS cells can be removed from the essential partof the plasmid for cDNA expression by this procedure. One to three daysculture under conventional culture conditions after transfection of thisvector to monkey cultures cell COS-7 (Gluzman, Y. Cell, vol. 23,175-182, (1981)), IL-2 is usually secreted and produced in cultured cellmedium. In order to insert amplified DNA into other eucaryotic cells,similarly a vector appropriate to host organisms is connected to cDNAinsert cleaved and isoalted from procaryotic cells and the eucaryoticcell may be transfected with thus synthesized vector and cultured.

Cells incorporating the recombinant DNA are cultured to amplify therecombinant DNA or to produce IL-2 polypeptide. The cultivation iscarried out by conventional means. For instance, transformed yeast maybe cultured in a medium containing source of carbon, a nitrogen source,inorganic salts and, when required, organic nutrients such as vitaminand amino acid at a temperature in the range from 20° to 37° C., and apH ranging from 4 to 7 under aerobic condition. Transformed procaryoticorganisms, such as E. coli or B. subtilis may also be cultured underconventional conditions.

The IL-2 produced intracellularly or extracellulary is recovered by anyknown method, such as precipitation with ammonium sulfate, dialysis toremove salts (under normal or vacuum pressure), gel filtration,chromatography, preparative flat-bed iso-electric focusing, gelelectropheresis, high performance liquid chromatography (hereinafter"HPLC"), (ion exchange, gel filtrtion and reverse phase chromatography),and affinity chromatography on dye bound carrier, on activated Sepharose4B coupled with monoclonal antibody against said IL-2 or on lectin boundSepharose 4B and the like. Methods of recovery, and purification ofIL-2, are described in Watson et. al., J. Exp. Med., 150, 849-861(1979), Gillis et. al., J. Immunol., 124, 1954-1962, (1980), Mochizukiet. al., J. Immunol Methods 39, 185-201, (1980), and Welte, K. et. al.,J. Exp. Med., 156, 454-464 (1982).

The polypeptide thus obtained shows the same biochemical and biologicalbehavior as has been known for IL-2 produced by mammalian cells bymitogen stimulation, and has IL-2 activity. The molecular weight isaround 15,000 dalton and IL-2 activity was completely neutralized orprecipitated with monoclonal anti-IL-2 antibody in the presence orabsence of immunoadsorbents, such as Igsorb (Enzyme Center). Inimmunoelectrophoresis, the IL-2 polypeptide shows only a singleprecipitate against the corresponding anti-IL-2 antibody. The IL-2activity remains stable after reduction with 2-mercaptoethanol, and isresistant to treatment with DNAse and RNAse as well as to heat treatmentat 56° C. for 30 min.. The activity is stable at a pH between pH 2 to 9.The IL-2 produced could promote the growth of monoclonal functional Tcells (cytotoxic T lymphocyte), enhance the thymocyte mitogenesis, giverise to the generation of anti-tumor specific cytotoxic T lymphocytesfrom memory state in the absence of the antigen, and could be used toaugment natural killer cell activity against YAC-1 and RLo1 cells.

Having now generally described this invention, the same will becomebetter understood by reference to certain specific examples which areincluded herein for purpose of illustration only and are not intended tobe limiting unless otherwise specified.

EXAMPLE 1

(1) Human T leukemia cell line, Jurkat cells (freely available in Japan,W. Germany and United States) were suspended in RPMI 1640 mediumcontaining 10 vol/vol % FCS and were irradiated with X-ray till 10,000roentgen at a room temperature for 50 seconds using X-ray irradiationapparatus Exs 150/300-4 (Toshiba, Japan), and thereafter the irradiatedcell was cultured for 5 days at 37° C. in 5% CO₂ incubator at a initialcell density of 1×10⁵ cells/ml in the culture medium mentioned above.The mutated cells (0.2 cells/well) were placed in wells 10 pieces offlat-bottomed microplates having 96 wells, and cultured at 37° C. in 5%CO₂ incubator for 21 days.

Clones obtained from the wells showing growth were repeatedlytransferred into fresh culture medium to propagate the clone sizes, andthe propagated clones were cultured for 24 hrs at a initial cell densityof 1×10⁶ cells/ml in the presence of 50 μg/ml of Con A and IL-2 activitywas measured according to the methods described before. Consequently ahuman T cell line designated as Jurkat-111 (hereinafter "J-111") (ATCCCRL8129), cloned from parent Jurkat, was selected, of which productivityof IL-2 was increased 40 times as much as that of the parent strain. Thecloned cell line J-111 could grow under conventional conditions and thegrowth rate shows almost the same with ordinary Jurkat cells.

(2) Cells (1×10⁵ /ml) of J-111 were inoculated in 1,000 ml of serum freesynthetic culture medium RITC 55-9 (Sato, T. et al., Exp. Cell Res.,138, 127-134, (1982)) in roller culture bottles (Falcon 3027) andcultured for 4 days at 37° C., and cells propagated were harvested bycentrifugation. The harvested cells were again inoculated in the mediummentioned above which had been added with 25 μg/ml of Con A to contain4×10⁶ cells/ml. In four batches of roller culture bottles (Falcon),1,000 ml of the inoculated culture medium was placed into each batch.The cultivation was continued for 6 hours with rotating.

(3) Jurkat cells (1.2×10⁶) thus stimulated with 25 μg/ml of Con A for 6hrs were suspended in 8,000 ml of phosphate buffer balanced with saline(hereinafter "PBS"). The cells were washed twice by centrifugation andwere resuspended in 800 ml of RSB solution (10 mM Tris-HCl, pH 7.5, 10mM NaCl, 1.5 mM MgCl₂) containing Ribonucleosides-Vanadyl Complex (10mM), an inhibitor of nuclease. Then a detergent NP-40 was added tocontain 0.05% as final concentration, followed by gentle mixing and thecell nuclei were removed by centrifugation for five minutes at 3,000 rpmat 4° C. SDS (0.5%) and EDTA (5 mM) were added to the supernatant andcytoplasmic RNA was extracted by addition of equal volume of phenol.After three times extraction with phenol, RNA was precipitated with twotimes volume of ethanol and precipitates were collected bycentrifugation, which were solubilized in 10 mM Tris-HCl of pH 7.5. Theamount of RNA obtained was 196 mg.

Fractionation of mRNA was carried out using affinity chromatography onoligo (dT)-Cellulose (P. L. Biochemicals, Type 7). An adsorptionsolution was a solution of pH 7.5 containing 20 mM Tris-HCl, 0.5M NaCl,1 mM EDTA and 0.5% SDS and elution was carried out with water and 10 mMTris-HCl (pH 7.5) by turns after washing the column with the buffer (20mM Tris-HCl pH 7.5, 0.5M NaCl, 1 mM EDTA). The resultant mRNA eluted was3.6 mg. Next, 2.4 mg of the mRNA obtained was fractionated by sucrosedensity gradient centrifugation (5 to 2.5% sucrose density gradient in asolution of pH 7.5 containing 50 mM Tris-HC, 1 mM EDTA and 0.2M NaCl,centrifuged at 26,000 rpm for 24 hrs at 4° C., and 11 to 12S fraction ofmRNA was fractionated into fractions No. 12, 13, 14 in the amount of 59μg, 46 μg and 60 μg, respectively.

(4) The mRNA obtained in fraction No. 13 was microinjected into theoocyte of Xenopus laevis (50 ng mRNA/egg) and the culture supernatantwas served for the assay of IL-2 activity. As shown in Table 1, theincrease of the incorporation of ³ H-TdR and the increase of number ofactivated T lymphocytes were confirmed, clearly verifying that mRNA inthis fraction contains human IL-2 mRNA.

                  TABLE 1                                                         ______________________________________                                        (a)                                                                                               Uptake of .sup.3 H-TdR                                                                     Amount of IL-2*                              Sample      Dilution                                                                              (cpm)        (unit/ml)                                    ______________________________________                                        Control I   --      553          0                                            (Medium for assay)                                                            Control II  ×2                                                                              590          0                                            (Supernatant of egg                                                                       ×32                                                                             572                                                       culture non-treated)                                                          Translation product                                                                       ×8                                                                              14,683       32                                           of fraction 13                                                                            ×32                                                                             10,165                                                    ______________________________________                                        (b)                                                                                               Cell number of                                                                T-lymphocyte Amount of IL-2*                                          Dilution                                                                              (No./well)   (unit/ml)                                    ______________________________________                                        Control I   ×2                                                                              0            0                                            (Medium for assay)                                                                        ×16                                                                             0                                                         Control II  ×2                                                                              0            0                                            (Supernatant of egg                                                                       ×16                                                                             0                                                         culture non-treated)                                                          Translation product                                                                       ×2                                                                              115          40                                           of fraction 13                                                                            ×16                                                                             55                                                        ______________________________________                                         *The unit was calculated by comparing the amount of incorporated .sup.3       HTdR with that of standard IL2 (10 unit/ml) according to probit analysis.

(5) Thereafter cDNA was synthesized in vitro from No. 13 fraction of 11to 12S mRNA containing IL-2 mRNA and recombinant DNA was constructedwith the plasmid vector PBR 322. With the recombinant DNA, Escherichiacoli was transformed, and clone acquired IL-2 cDNA clones was selected,as follows:

(5-1) Fifty mM Tris-HCl buffer (pH 7.5) , 30 mM NaCl, 6 mM MgCl₂, 5 mMdithiothreitol (hereinafter "DTT"), 0.5 mM of each dATP, dGTP, dCTP,dTTP (dCTP contained ³² P radiolabelled one), 0.7 μg oligo (dT)₁₀ , 10μg mRNA and 15 unit AMV reverse transcriptidase (J. W. Beard) were mixedand maintained for 90 min. at 41° C.

After termination of the reaction, DNA was recovered as ethanolprecipitates after the phenol treatment, and DNA was solubilized in asolution of pH 7.5 containing 20 mM Tris and 1 mM EDTA.

Two point five μg of ss-cDNA was synthesized. To remove mRNA present inthis solution, the solution was made 0.33 N-NaOH by addition of NaOH,allowed to stand for 15 hrs at a room temperature, then the solution wasneutralized with equal volume of 1M-Tris-HCl of pH 7.5 and passedthrough "Sephadex G-50" column. The recovered cDNA was 1.8 μg.

(5-2) Fifty mM phosphate buffer (pH 7.5), 10 mM MgCl₂, 10 mM DTT, 0.75mM of each dATP, dGTP, dCTP, dTTP (dCTP contains ³ H radiolabelled one),1.8 μg ss-cDNA, and 8 unit of polymerase I (BR6, United States) weremixed and were allowed to react for 15 hrs at 15° C. After thetermination of the reaction, DNA was recovered as ethanol precipitate,after treatments with phenol and with chloroform. 1.10 μg of ds-cDNA wasgenerated. A mixture of 50 mM sodium acetate (pH 4.5), 0.2M NaCl, 1 mMZnCl₂ and 1.10 μg of ds-cDNA was incubated for 20 min. at 37° C., addedwith 0.25 unit of nuclease S₁ (Sankyo, Japan), and incubated further for15 min.

After the termination of the reaction, the reaction product treatedtwice with phenol was applied onto sephadex G-50 to get 0.55 μg ofds-cDNA.

(5-3) A mixture of 0.14M potassium cacodylate, 30 mM Tris base, 0.1 mMDTT, 1 mM COCl₂, 0.64 mM ³² P-dCTP (spc. act. 2.7×10⁶ cpm/n mol), 0.55μg of ds-cDNA and 5 unit of termninal transferase (BRL) were incubatedfor 7 min. at 37° C., then applied onto sephadex G-50 column afterphenol treatment to get 0.50 μg DNA as ethanol precipitates. Therecovered DNA was found to be extended with around 50 dCMP residues atthe both 3' terminus.

Ten λg of pBR 322 DNA was cleaved with restriction enzyme PstI, and3'-termini of the cleaved DNA were added with dGMP chain, by the samemethod as that used in the addition of dCMP to ds-cDNA mentioned above,except dGTP was used in place of dCTP.

(5-4) A mixture of 50 mM Tris-HCl (pH 7.5), 0.1M NaCl, 5 mM EDTA, 0.05μg of pBR 322 elongated with dGMP residues and 0.01 μg of cDNA extendedwith dCMP was incubated firstly for 2 min. at 65° C., then for 120 min.at 46° C., for 60 min. at 37° C. and finally for 60 min. at a roomtemperature.

E. coli χ 1776 (Curtiss III, R. et al., in Molecular Cloning oRecombinant DNA, (W. A. Scott & R. Werner ed.) Academic Press, (1977))was inoculated in 50 ml of L broth containing 100 μg/ml ofdiaminopimelic acid, 50 μg/ml of thymidine, 1% tryptophan, 0.5% yeastextract, 0.5% NaCl and 0.1% glucose and cultured in shaking at 37° C.until the absorbance of culture liquid at 562 nm became around O. D 0.3.After the termination of the culture, the culture liquid was left at 0°C. for 30 min., then the bacterial cells were collected bycentrifugation followed by twice washing with 25 ml of a solutioncontaining 5 mM Tris-HCl (pH 7.6), 0.1M NaCl, 5 mM MgCl₂ and 10 mM RbCl.

Thus obtained cells were suspended in 20 ml of a solution containing 5mM Tris-HCl (pH 7.6), 0.25M KCl, 5 mM MgCl₂, 0.1M CaCl₂ and 10 mM RbCland were left at 0° C. for 25 min., then cells were collected toresuspend them into 1 ml of the same solution, the recombinant DNAdescribed above was added into 0.2 ml of the cell suspension and thesuspension was left at 0° C. for 60 min. Then 0.7 ml of L broth wasadded to culture in shaking for 30 min. at 37° C. Thus obtained culturemedium (0.1 ml) was thoroughly spread on the surface of 1.5% agarosemedium composed of L broth containing 100 μg/ml diamonopimelic acid, 50μg/ml thymidine and 15 μg/ml tetracycline, and incubated at 37° C. fortwo days.

(5-5) Four hundred and thirty two colonies appeared were divided into 18groups, each containing 24 different bacterial clones, inoculated in 200ml of L-broth containing 100 μg/ml of diaminopimelic acid, 50 μg/ml ofthymidine and 10 μg/ml of tetracycline and cultured in shaking at 37° C.for 5 to 7 hrs. Then, 200 ml of fresh L-broth containingchloramaphenicol at a final concentration of 170 μg/ml was added toculture further for an overnight. Thus amplified plasmid DNA waspurified according to a conventional mean. Clones possessing IL-2 cDNAwere screened by a mRNA hybridization-translation assay (hereinafter"H-T" assay"). H-T assay here employed was carried out as follows:Purified DNA (25 μg) was cleaved with restriction enzyme Hind III,treated with phenol three times, treated with phenol-choroform and withchloroform, respectively, precipitated with ethanol, washed with 80%ethanol and dissolved in 40 μl of 80% formamide.

The reaction mixture was heated for denaturation at 90° C. for 5 min.,then diluted to 1.3 ml with 10×SSC (1.5M NaCl, 0.15M sodium citrate).The DNA was thereafter fixed onto nitrocellulose filters, which filterswere dried up at 80° C. for 3 hrs. and incubated for 18 hrs at 37° C. inthe solution containing 50% formamide, 20 mM Pepes of pH 6.5, 0.75MNaCl, 5 mM EDTA, 0.2% SDS and 250 μg of poly (A) mRNA from induced J-111cells to hybridize the DNA fixed on filters with IL-2 mRNA. Then thefilters were washed at 65° C. three times with solution consisting of 10mM Pipes of pH 6.5, 0.15M NaCl, 1 mM Pipes, 10 mM NaCl solution andtreated with 0.5 mM EDTA, 0.1% SDS solution at 95° C. for 1 min. torecover the hybridized mRNA from the filters. Thus extracted mRNA waspurified on oligo dT-Cellulose column according to the conventionalmethods and injected into Xenopus oocytes to determine the IL-2 activityof translated proteins. One out of 18 groups, each consisting of 24clones, gave positive 48 unit/ml IL-2 activity in ³ H-TdR incorporationassay described previously, while others being clearly negative. Then 24single colonies belonging to the positive group were inoculated in 200ml of L-broth possessing the same composition described, culturedaerobically for 5 to 7 hrs. at 37° C. and similarly chloramphenicolcontaining fresh L-broth was further added. After amplification ofplasmid DNA by an overnight culture, plasmid DNA was similarly purifiedaccording to the standard procedures. After cleavage of about 5 μg ofeach plasmid DNA with Hind III, each plasmid DNA was bound tonitrocellulose filters similarly. The filters were hybridized with IL-2mRNA and hybridized mRNA was recovered to inject into Xenopus oocyte todetermine the IL-2 activity of translated proteins.

As shown in Table 2, only plasmid DNA purified from a single colony,designated as p3-16, gave the positive IL-2 activity. Therefore thisclone was identified as the clone possessing IL-2 cDNA (E. coli χ1776/p3-16 AJ 11995 (FERM-BP-225)). Thus plasimid DNA, p3-16, wasconfirmed to share exactly the DNA (I1-2 gene) capable of forming thespecific hybrid with IL-2 mRNA.

                  TABLE 2                                                         ______________________________________                                        (a)                                                                                               Uptake of .sup.3 H-TdR                                                                     Amount of IL-2                               Sample      Dilution                                                                              (cpm)        (unit/ml)                                    ______________________________________                                        Control I   --      2,010        0                                            (Medium for assay)                                                            Control II  ×2                                                                              2,120                                                     (Supernatant of                                                                           ×32                                                                             2,482        0                                            culture liquid of                                                             non-treated egg)                                                              Translation product                                                                       ×2                                                                              20,453       58                                           of mRNA     ×32                                                                             20,961                                                    ______________________________________                                        (b)                                                                                               Cell number of                                                                T lymphocyte Amount of IL-2                               Sample      Dilution                                                                              (cells/well) (unit/ml)                                    ______________________________________                                        Control I   --      0            0                                            (Medium for assay)                                                            Control II  ×2                                                                              0            0                                            (Supernatant of                                                                           ×32                                                         culture liquid of                                                             non-treated egg)                                                              Translation product                                                                       ×2                                                                              88           32                                           of mRNA*    ×32                                                                             42                                                        ______________________________________                                         *mRNA hybridized with cDNA frorn plasmid p3-16.                          

The cDNA insert of plasmid p3-16 showed characteristics to be cleaved byrestriction enzyme XbaI at a single site and by BstNI at two sites, (atupstream and downstream of XbaI cleavage site). However the plasmidp3-16 contained a cDNA insert consisting of about 650 base pairs, whichapparently corresponds to a part of IL-2 mRNA of 11 to 12S size.

Therefore another cDNA library were prepared according to the procedureof Land et al. (Land et al., Nucleic Acids Res., vol 9, p2551, (1981))using IL-2 mRNA as a template. Single stranded cDNA (1.6 μg) wassynthesized by using 4 μg of IL-2 MRNA elongated by dCMP residues, andds-cDNA was synthesized by using oligo (dG) ₁₂₋₁₈ as the primer for DNApolymerase I (Klenow fragment). The cDNA (0.6 μg) longer than 680-basepair DNA size marker was obtained by a sucrose gradient centrifugationand inserted into the PstI site of pBR322 by the standard G-C tailingmethod. After transformation of E. coli χ 1776 by the recombinant DNA,approximately 2,000 colonies were screened by in situ hybridizationmethod of Grunstein-Hogness with nick-translated p3-16 cDNA insert asthe probe and the colony containing plasmid pIL 2-50A containing around850 base pairs and the transformed clone (E. coli χ 1776/pIL 2-50A, AJ11996 (FERM-BP-226)) were identified. A restriction endonucleasecleavage maps of the cDNA insert of pIL 2-50A are shown in FIG. 1. Toisolate a gene coding for IL-2 peptide from transformed E. coli. χ 1776pIL 2-50A, plasmid DNA was digested with restriction enzyme PstI afterisolation of DNA region from the cells according to the conventionalmeans. Thus produced smaller fragment among generated two DNA fragmentswas DNA gene coding for IL-2 peptide. The complete nucleotide sequenceof the PstI insert from pIL 2-50A was determined by the procedure ofMaxam and Gilbert (Maxam, A. W. et al., Enzym. 65, 499-1980), and thewhole structure is shown in FIG. 2.

EXAMPLE 2

The plasmid pKCR (O'Hare et al. Proc. Natl. Acad, Sci., U.S.A., vol 78,No. 3, 1527-1531, (1981)) consists of (i) segments of SV40 DNA (shown ashatched blocks in FIG. 3) containing an early gene promoter and anorigin of replication (0.725-0.648 m.u.) and a polyadenylation site fromthe early gene (0.169-0.144 m.u.) (ii) a part of the rabbit β-globingene (shown as open blocks) (BamHI-PvuII fragment) (iii) a segment frompBR322 (EcoRI-BamHI fragment) containing an origin of replication andampicillin resistance gene. This plasmid was cleaved by BamHI, and,after filling both ends of the cleaved DNA by DNA polymerase I (Klenowfragment), a synthetic PstI linker DNA was introduced to construct pKCR(PstI). Plasmid pKCR (PstI) was cleaved by SalI, treated by the Klenowfragment to fill the ends and then partially cleaved by EcoRI to obtainEcoRI-SalI fragment which contains the whole DNA derived from SV40 andthe globin gene. This fragment was then ligated to a piece of pBR328 DNAwhich contains tetracycline resistance gene and an origin of replicationas outlined in the FIG. 3. The resulting plasmid pCE-1 contains a singlePstI site just downstream of the SV40 early promoter.

The cDNA insert of pIL 2-50A was excised by PstI cleavage and ligated toPstI-cleaved pCE-1 to construct pCEIL-2 in which expression of the IL-2structural gene should be under control of SV40 early promoter. PlasmidpCE-1 was originally constructed for the cDNA cloning by G-C tailingmethod (Chang, A. C. Y. et al. Nature, 275, 617-624, 1978) in bacteriaand direct expression in mammalina cells.

This plasmid was digested by HhaI and then introduced by DNAtransfection (McCutchan et al., J. Natl. Cancer Inst. 41, 351-357, 1968)into the transformed monkey cell line COS-7 which allows replication ofDNA containing SV40 origin sequences and is available from Gluzman,Y.(Cell, 23, 175-182, 1981). It appears to be important to digest theplasmid by HhaI before transfection for the efficient expression of cDNAsince sequences which could hamper replication of the transfected DNA inCOS cells can be removed from the essential part of the plasmid for cDNAexpression by this procedure. COS-7 cells (6×10⁴ /ml) were suspended in0.5 ml of DMEM containing 5% FCS in 24 well culture plate (Nunc) andincubated for 4 hrs. at 37° C. Then mixture of 1 μg of the abovedescribed vector, 17.6 μl of 1 mM Tris-HCl containing 0.1 mM EDTA, 2.4μl of 2M CaCl₂ and 120 μl of 2×HBS containing 50 mM Pipes, 280 mM NaCl,and 1.5 mM Na₂ HPO₄.12H₂ O (pH 7.10) were added to the cultured cells.The cells were further incubated for 4 hrs. at 37° C. and the culturemedium was aspirated off, washed with 1 ml of PBS, then 0.5 ml of PBScontaining 20% glycerol was added to leave at a room temperature for 3min. Again the medium was aspirated off and the cells were washed with 1ml of PBS and cultured in 1 ml of DMEM containing 5% FCS. Every 24 hrs.,500 μl of medium was exchanged with fresh medium. Each media, collectedat appropriate interval was kept at 4° C. until use. Two to three daysafter the transfection, the cultured cell medium was assayed for thehuman IL-2 activity. As shown in Table 3 the resulting culturesupernatant of COS-7 cell transfected with PCEIL-2 contained IL-2activity. No IL-2 activity was detectable in the culture media of cellstransfected with pCE-1.

                  TABLE 3                                                         ______________________________________                                                   IL-2 activity measured                                             DNA with which                                                                           by .sup.3 H-TdR uptake                                                                      Growth of T-lympho-                                  transfected                                                                              (U/ml)        cyte                                                 ______________________________________                                        PCEIL-2    12            ++++                                                 pCE-1      1             -                                                    ______________________________________                                    

The IL-2 activity found in culture cell medium after transfection ofCOS-7 with pCEIL-2 was neutralized from 12 unit/ml to below 1 unit/ml bymouse (BALB/c) anti-human IL-2 monoclonal actibody. The result thatCOS-7 cell transfected with pCEIL-2 secreted human IL-2 clearly showsthat cells of eukaryote transformed with a recombinant DNA comprising agene coding for IL-2 polypeptide and a vector DNA capable of replicatingin said cells can exactly useful for the production of IL-2.

The plasmid PCEIL-2 incorporated in E. coli. HB101 has been deposited inthe accession number of FERM-BP 244.

EXAMPLE 3

Escherichia coli χ 1776/pIL 2-50A (AJ 11996 (FERM-BP-226)) prepared inExample 1 was inoculated in 250 ml of L broth, containing 100 μg/mldiaminopimelic acid, 50 μg/ml thymidine, 1% tryptophan, 0.5% yeastextracts, 0.5% NaCl and 0.1% glucose, and cultured with shaking at 37°C. till optical density at 562 nm of the cultured medium became 0.5.After the termination of the culture, cultured medium was allowed tostand at 0° C. for 30 min. and the cells were harvested bycentrifugation, washed once with 20 mM Tris-HCl containing 30 mM NaCland were resuspended in 1.8 ml of the same buffer. A solution containing0.2 μl of lysozyme (10 mg/ml) and 20 μl of 0.5M EDTA was then added tothe cells and the mixture was allowed to stand at 0° C. for 20 min.,followed by freeze-thawing three times successively. Then extracts ofcells (1.5 ml) were obtained after centrifugation 40,000 rpm for 30 min.The extract was subjected to salting out with 85% ammonium sulfates,applied on sephadex G15 to remove salts, then applied on DEAE cellulosecolumn chromatography and the fraction eluted with 0.06M Tris-HCl buffer(pH 7.6) was pooled. Thus pooled fraction was freeze-dried and wasapplied on controlled pore glass beads (250Å, Funakoshi pharmaceuticals,Japan) chromatography to get IL-2 activity in eluant with 0.3Mglycine-HCl buffer, where IL-2 containing fraction exerted 12 unit/ml ofIL-2 activity. The results clearly indicate that E. coli. χ 1776/pIL2-50A, AJ 11996 actually produces IL-2.

EXAMPLE 4

Constitutive IL-2 producer cell line J-A1886 (ATCC CRL3130), cloned fromJurkat cells according to the means described in Example 1, wassimilarly grown in roller culture bottle. The grown cells wereresuspended in fresh synthetic medium RITC-55-9 at a initial celldensity of 1×10⁶ cells/ml and 8 hrs. after the start of the culture, thecells were served for the extraction of IL-2 MRNA as 11 to 12S fraction,from 3×10⁹ cells, according to the steps detailed in Example 1.

Double stranded cDNA was synthesized similarly as Example 1 and the cDNAlonger than 600 base pairs (2.4 μg) was obtained after fractionation ona sucrose density gradient. The cDNA was then extended with dCMPresidues using terminal deoxynucleotidyl transferase and an aliquot (50ng) was annealed with 250 ng of dGIP-elongated, PstI-cleaved pBR322. Theresulting hybrid plasmids were used to transform E. coli. χ 1776 and thetransformants of around 4,000 clones were obtained. According to theGvunstein-Hogness method, three clones complementary with plasmid 3-16cDNA used as a probe were selected. Namely thus selected clones aretransformed clones possessing human IL-2 gene.

EXAMPLE 5

A plasmid which should direct the synthesis of human IL-2 in E. coli.cells was constructed as follows. A plasmid pTIL2-22 was constructedfrom pTrS-3 (Nishi T., Taniguchi T. et al., SEIKAGAKU 53, 967, (1981)),and pIL 2-50A containing the IL-Z cDNA by a series of modificationprocedures as illustrated in FIG. 5(a). A plasmid pTrS-3 includeinsertion of the region of Trp promoter and Shine Dalgarno (hereinafter"SD") between EcoRI site and ClaI site of pBR322. The plasmid alsocontains an ATG initiation codon 13 bp downstream of the SD sequence aswell as a single SphI site as illustrated in FIG. 4. The vector is veryefficient to produce the said protein when DNA sequence corresponding tothe said protein is inserted in phase just downstream of the ATG codon,which is generated by SphI digestion and by subsequent treatment by T4DNA polymerase of pTrS-3. Therefore the plasmid pTrS-3 (30 μg) wascleaved with a restriction enzyme SphI in a conventional manner andafter successive treatment with phenol and chloroform, ethanolprecipitates were recovered, then both ends were rendered flush by thetreatment of T4 DNA polymerase. Then the DNA (21.4 μg) was recovered bysimilar successive phenol, chloroform treatment and ethanolprecipitation. On the other side, 380 μg of pIL 2-50A containing an IL-2cDNA was cleaved by PstI and the IL-2 cDNA insert was isolated byagarose gel electrophoresis. cDNA insert (11 μg) was cleaved by HgiAI,treated by T4 DNA polymerase and 10 μg of the DNA of larger site wasisolated by agarose gel electrophoresis. According to the procedures acDNA (7.2 μg) coding for 132 amino acids was obtained and this DNAfragment had blunt ends (FIG. 5(a)). Then the thus obtained cDNAfragment ligated to a pTrS-3 vector, previously digested by SphI andtreated by T4 DNA polymerase just downstream of ATC sequence. Thusligated plasmid was then used to transform into E. coli. HB101 accordingto the conventional procedures. Ligation was carried out as follows.IL-2 cDNA (0.4 μg) larger fragment and 0.2 μg of pTrS-3 vector DNA weremixed with 0.8 unit of T4 DNA ligase in 66 mM Tris-HCl of pH 7.5containing 6.6 mM MgCl₂, 1 mM ATP and 10 mM DTT, and the mixture wasallowed to react at 4° C. overnight. Among the transformants appeared onL broth agar plate containing ampicillin, colonies containing the IL-2cDNA portion, which encodes 132 amino acids were selected by in situcolony hybridization assay. Thus selected colonies were cultured (10 ml)again to prepare plasmid DNA by lysozyme treatment and byfreeze-thawing. The plasmid DNAs were cleaved with PstI and XbaI, andthe resulting products were analysed by agarose gel electrophoresis inorder to identify pTIL 2-22 in which the cDNA was linked to the ATGsequence of pTrS-3 in correct orientation. The E. coli. HB101 containingPTIL 2-22 was cultured under the conventional conditions known for thepropagation of microorganisms. The cells were grown in 10 ml of χ broth(2.5% Bactotrypton, 1.0% yeast extracts, 0.1% glucose, 20 mM MgSO₄, 50mM Tris-HCl, pH 7.5) containing 25 μg/ml streptomycin and 25 μg ofampicillin at 37° C. for an overnight. One ml of the culture suspensionwas inoculated into the same χ broth (100 ml) and cultured at 37° C.When O.D at 650 mμ arrived around 1.5-2.0, 3-indole acrylic acid (IAA)was added. Three hours after the addition of inducer, the cells werecollected, washed with 20 mM Tris-HCl (pH 7.5, 30 mM NaCl) andresuspended into 8 ml of the same buffer. For the efficient functioningof Trp promoter inducers such as IAA was added at a final concentrationof 50 μg/ml. Thus produced proteins in bacterial cells were extracted bysonication (0° C. 2 min.) or lysozyme (8 μg) digestion (0° C., 20 min.)followed with three successive freeze-thawing. According to thisprocedures IL-2 was usually extracted from organisms. The extracted IL-2activity ranged from 10,000 to 12,000 units/ml.

E. coli. HB101 containing pTIT 2-22 has been deposited in the accessionnumber of FERM-BP 245.

EXAMPLE 6

A plasmid pTuIL 2-22, carrying IL-2 cDNA, was constructed from pTuBlP-5(Taniguchi, T. et al., Seikagaku, 53, 966, 1981) and pTIL 2-22 shown inExample 5, by the procedures as illustrated in FIG. 7. A plasmidpTuBlP-5 includes insertion of the promoter sequence for tufB in PBR322.The plasmid also contains a single ClaI site and this is located 2bPdownstream of the SD sequence as shown in FIG. 7. Since pTrS-3 alsocontains a ClaI site between the SD sequence and ATG intitiation codon,and since this ClaI site is not destroyed during the construction ofexpression plasmid by using pTrS-3 IL-2 cDNA as described in Example 5,it is very simple to replace the bacterial trp promoter with that oftufB so that the IL-2 cDNA is expressed under the control of tufBpromoter.

Therefore the plasmid pTIL 2-22 (30 μg) was cleaved with a restrictionenzyme ClaI and PvuII in a conventional manner. The fragment (ca 2.2 kb)containing IL-2 cDNA was isolated and purified by agarose gelelectrophoresis to recover 3 μg of DNA. On the other side, 20 μg ofpTuBIP-5 vector was cleaved similarly by ClaI and PvuII, and the largerfragment (ca. 3.4 kb) containing ampicillin resistant gene was isolatedand purified by agarose gel electrophoresis to recover 3.5 μg of DNA.Then thus obtained two fragments, one (ca. 3.4 kb) containing tufBpromoter, the other (ca. 2.2 kb) containing IL-2 cDNA, were ligated asfollows. The fragment containing IL-2 cDNA (1.2 μg) and 0.3 μg of thefragment containing tufB promoter were mixed with 0.8 unit of T4 DNAligase in 66 mM Tris-HCl of pH 7.5 containing 6.6 mM MgCl₂, 1 mM ATP and10 mM DTT, and the mixture was allowed to react at 4° C. overnight. Thusligated plasmid was then used to transform into E. coli HB101 accordingto the conventional procedures. Among the transformants appeared onLbroth agar plate containing ampicillin, eight colonies containing theIL-2 cDNA portion such as pTuIL 2-22 in FIG. 7 were selected and plasmidDNA was prepared as described in Example 5. The E. coli HB101 containingpTuIL 2-22 were cultured in L broth (100 ml) at 37° C. When O.D at 650mμ arrived around 0.5-1.0, the bacterial cells were collected, washedwith 20 mM Tris-HCl (pH 7.5, 30 mM NaCl) and resuspended into 2 ml ofthe same buffer. Thus produced proteins were extracted similarly asExample 5. The extracted IL-2 activity ranged from 6,000 to 56,000units/ml.

Escherichia coli HB101 containing pTuIL 2-22 has been deposited as inthe accession number of FERM-246.

EXAMPLE 7

A plasmid pGIL 2-22, carrying IL-2 cDNA was constructed from pGL 101(Roberts, T. M. and Laucer G. D., Meth. Enzym., 68, 473-483, 1979) andpTIL 2-22 shown in Example 5.

The plasmid pGL 101 (20 μg) containing a lac promoter was cleaved with arestriction enzyme PvuII in a conventional manner to recover 17 μg ofDNA by successive treatment with phenol, chloroform and ethanolprecipitation. On the other side, pTIL 2-22 (75 μg) was cleaved withClaI and SalI to recover 2.2 μg of a DNA fragment containing IL-2 cDNAby agarose gel electrophoresis. The fragment was rendered flush by thetreatment with DNA polymerase I (Klenow fragment), then thus obtainedtwo fragments (0.25 μg and 0.66 μg) were ligated with 1.0 unit of T4 DNAligase in the same manner as Example 6. Thus ligated plasmid was thenused to transform E. coli HB101 according to the conventional manner.Among the transformants, the transformants possessing the insertion ofthe ClaI-SalI fragment containing IL-2 cDNA as a probe. Thesetransformants were then cultured in χ broth (10 ml) containing 25 μg/mlof ampicillin and the plasmid DNA was prepared by the manner asdescribed in Example 5. Thus the plasmid DNA possessing the initiationsequence ATG of IL-2 cDNA just downstream of a lac promoter was obtainedby cleavage with PstI and XbaI.

Thus prepared pGIL 2-22 was inoculated in 100 ml of L-broth containing25 μg/ml of ampicillin and 25 μg/ml of streptomycin and were cultured.When optical density at 650 mμ arrived around 0.5,isopropyl-β-D-thiogalactopyranoside (IPTG) was added in theconcentration of 1 mM and one hour later the bacterial cells werecollected and the cell extracts were prepared in the manner as describedin Example 6. The extracted IL-2 activity ranged from 6,000 to 80,000units/ml.

Escherichia coli HB101 containing pGIL 2-22 has been deposited in theaccession number of FERM-BP 247.

EXAMPLE 8

Plasmid pTrS-3 (10 μg) was at first cleaved with the restriction enzymeSalI and the SalI site was rendered flush by the treatment with DNApolymerase (Klenow fragment) or with T4 DNA polymerase. After cleavagewith ClaI, a larger fragment, containing the trp promoter region, wasisolated by agarose gel electrophoresis in a conventional manner torecover 3 μg of DNA.

On the other side, 11 μg of cDNA insert obtained by the PstI cleavage ofpIL2-50A was cleaved with HgiAI, treated with T4 DNA polymerase and alarger fragment was isolated and purified by agarose gelelectrophoresis. Thus cDNA fragment coding for 132 amino acids of IL-2was obtained in an amount of 7.2 μg. Then 0.45 μg of the fragmentcontaining a trp promoter (described above), 0.5 μg of HgiAI-PstIfragment containing IL-2 cDNA and synthetic oligonucleotides (5')CGATAAGC TATGGCA (3'), and (3') TATTCGATACCGT (5') (each 20 pmole), bothof which were phosphorylated at 5'-terminus, were ligated with 1 unit ofT4 DNA ligase in the same manner as described in Example 5.

Thus ligated plasmid was then used to transform E. coli HB101. Among thetransformants appeared, the target transformants were selected asfollows.

The candidate transformants able to hybridize with both of IL-2 cDNA andsynthetic oligonucleotides were firstly selected by colony hybridizationmethod, then the transformants possessing the insertion of DNA fragmentinitiating from CCT sequence at position III to 113 in FIG. 2(a)(CCTACT-----) just downstream of ATG GCA sequence were selected by PstI,XbaI cleavage.

The above transformant, which contains pTIL2-21a or pTIL2-21b, iscultured in L broth by the manner shown in Example 5, and highactivities of IL-2 can be found in cell extracts of the transformantswhen assayed by the manner shown in Example 5.

Escherichia coli HB101 possessing pTIL2-2.1a (AJ 12013) and Escherichiacoli HB101 possessing pTIL2-21b (AJ 12014) have been deposited in theassession numbers of FERM-BP 248 and FERM-BP 249 respectively.

The hosts, E. coli χ 1776 and HB101 (Boyer H. W. et al., J. Mol. Biol.41, 459, (1969)) used in the above Examples are known and available forany public. Additionally, the hosts can be obtained from the depositedtransformants by culturing the transformants in L-broth at 37° C. tomake release the respective recombinant DNAs in the transformants andseparating strains which become sensitive to tetracycline and ampicillinas the hosts.

The plasmid vectors pBR322 (Which is commercially sold by, for example,Bethesda Research Laboratory), pCE-1, pTrS-3 and pGL101 are known andavailable for any public. In addition, the plasmid vectors can beobtained from the deposited transformants by separating the recombinantplasmid DNAs in the transformants by a conventional manner and byseparating the plasmid vectors by the manners which are naturallyobvious from the disclosures in the respective Examples. For example,pCE-1 can be obtained by digesting pCEIL-2 by PstI and separating largerDNA fragment formed. Additionally, pTrS-3 and pTuBlP-5 have beendeposited as E. coli FERM-P 6735 and E. coli ATCC 31871 respectively.

Having now fully described this invention, it will be understood bythose of skill in the art that the same can be performed within a wideand equivalent range of conditions, parameters and the like withoutaffecting the spirit or scope of the invention or of any embodimentthereof.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A DNA vector comprising the DNA sequence offrom the adenine at nucleotide position number 52 to the nucleotide atposition number 542 of the DNA sequence of FIG. 2(a).
 2. A cellcomprising the DNA vector of claim
 1. 3. A DNA vector comprising a DNAsubsequence encoding from amino acid residue number three to amino acidresidue number 153 of the amino acid sequence of FIG. 2(a).
 4. A cellcomprising the DNA vector of claim 3.